Tailored high-throughput low-flow LC-MS methods for large sample cohort analysis

Importance of the topic

High-throughput low-flow LC-MS methods address critical needs in clinical and translational proteomics. They combine the sensitivity and multiplexing capacity of mass spectrometry with the robustness and speed required for large cohort studies. By optimizing analysis times and MS utilization, these methods enable comprehensive protein profiling of biofluids such as plasma or serum and support biomarker discovery, personalized medicine, and routine QA/QC workflows.

Objectives and study overview

This study presents a portfolio of five capillary-flow LC-MS methods with cycle times of 8, 14.4, 24, 48, and 60 minutes. The goals are to:

• Balance throughput and proteome depth for large clinical sample sets
• Ensure high MS utilization for maximum data yield
• Maintain chromatographic robustness without hardware changes

Methods and sample preparation

Serum samples underwent a simple workflow compatible with automation:

• Methanol precipitation of proteins from 50 µL serum
• Pellet digestion in a two-step protocol at 37 °C for 3 hours without reduction/alkylation
• Acid quench and C18 SPE cleanup (HyperSep C18 cartridge)
• Final reconstitution in 0.1% formic acid prior to LC-MS

Instrumental setup

The LC-MS platform comprised:

• Thermo Scientific™ UltiMate 3000 RSLCnano with ProFlow™ flow meter (50–1500 nL/min)
• EASY-Spray connection kit and ES800A column (3 µm, 75 µm × 15 cm)
• Orbitrap Exploris 480 or Q Exactive HF-X mass spectrometer for DDA acquisition

Main results and discussion

HeLa digest benchmarks (200 ng injections):

• 8 min method: ~5,900 peptide groups, ~1,155 proteins
• 48 min method: ~27,000 peptide groups, ~3,000 proteins

Crude serum digest profiling:

• 8 min: >1,000 peptides, ~138 proteins
• 60 min: ~2,748 peptides, ~265 proteins

Robustness test with 200 consecutive serum injections over 3 days showed stable retention times, peak shapes, and signal intensities.

Benefits and practical applications

These tailored methods offer:

• High MS utilization (>75–95%)
• Throughput of 24 to 180 samples per day
• Proteome coverage spanning five orders of magnitude in concentration
• No need for fluidic or hardware reconfiguration between deep-dive and high-throughput modes
• Minimal carryover and compatibility with complex matrices

Future trends and potential applications

Emerging directions include integration of spectral library searches and advanced peak detection algorithms to boost identification rates by up to 30%. Automation of sample preparation and multiplexed quantitative workflows will further enhance throughput. Adoption in personalized medicine, large-scale epidemiological studies, and regulatory environments is anticipated as instrumentation and software continue to evolve.

Conclusion

The described portfolio of high-throughput low-flow LC-MS methods delivers a flexible solution for large cohort proteomics. By tuning cycle times and flow rates without hardware changes, these approaches combine speed, depth, and robustness for diverse clinical research and QA/QC applications.